The essential zinc finger protein ASCIZ (also known as ATMIN, ZNF822) plays critical roles during lung organogenesis and B cell development in mice, where it regulates the expression of dynein light chain (DYNLL1/LC8), but its functions in other species including invertebrates are largely unknown. Here we report the identification of the Drosophila ortholog of ASCIZ (dASCIZ) and show that loss of dASCIZ function leads to pronounced mitotic delays with centrosome and spindle positioning defects during development, reminiscent of impaired dynein motor functions. Interestingly, similar mitotic and developmental defects were observed upon knockdown of the DYNLL/LC8-type dynein light chain Cutup (Ctp), and dASCIZ loss-of-function phenotypes could be suppressed by ectopic Ctp expression. Consistent with a genetic function of dASCIZ upstream of Ctp, we show that loss of dASCIZ led to reduced endogenous Ctp mRNA and protein levels and dramatically reduced Ctp-LacZ reporter gene activity in vivo, indicating that dASCIZ regulates development and mitosis as a Ctp transcription factor. We speculate that the more severe mitotic defects in the absence of ASCIZ in flies compared to mice may be due to redundancy with a second, ASCIZ-independent, Dynll2 gene in mammals in contrast to a single Ctp gene in Drosophila. Altogether, our data demonstrate that ASCIZ is an evolutionary highly conserved transcriptional regulator of dynein light-chain levels and a novel regulator of mitosis in flies.

The second most commonly mutated gene in primary microcephaly (MCPH) patients is wd40-repeat protein 62 (wdr62), but the relative contribution of WDR62 function to the growth of major brain lineages is unknown. Here, we use Drosophila models to dissect lineage-specific WDR62 function(s). Interestingly, although neural stem cell (neuroblast)-specific depletion of WDR62 significantly decreased neuroblast number, brain size was unchanged. In contrast, glial lineage-specific WDR62 depletion significantly decreased brain volume. Moreover, loss of function in glia not only decreased the glial population but also non-autonomously caused neuroblast loss. We further demonstrated that WDR62 controls brain growth through lineage-specific interactions with master mitotic signaling kinase, AURKA. Depletion of AURKA in neuroblasts drives brain overgrowth, which was suppressed by WDR62 co-depletion. In contrast, glial-specific depletion of AURKA significantly decreased brain volume, which was further decreased by WDR62 co-depletion. Thus, dissecting relative contributions of MCPH factors to individual neural lineages will be critical for understanding complex diseases such as microcephaly.

The Drosophila testis has been fundamental to understanding how stem cells interact with their endogenous microenvironment, or niche, to control organ growth in vivo. Here, we report the identification of two independent alleles for the highly conserved tumor suppressor gene, Retinoblastoma-family protein (Rbf), in a screen for testis phenotypes in X chromosome third-instar lethal alleles. Rbf mutant alleles exhibit overproliferation of spermatogonial cells, which is phenocopied by the molecularly characterized Rbf11 null allele. We demonstrate that Rbf promotes cell-cycle exit and differentiation of the somatic and germline stem cells of the testes. Intriguingly, depletion of Rbf specifically in the germline does not disrupt stem cell differentiation, rather Rbf loss of function in the somatic lineage drives overproliferation and differentiation defects in both lineages. Together our observations suggest that Rbf in the somatic lineage controls germline stem cell renewal and differentiation non-autonomously via essential roles in the microenvironment of the germline lineage.

Here, we report novel tumour suppressor activity for the Drosophila Argonaute family RNA-binding protein AGO1, a component of the miRNA-dependent RNA-induced silencing complex (RISC). The mechanism for growth inhibition does not, however, involve canonical roles as part of the RISC; rather, AGO1 controls cell and tissue growth by functioning as a direct transcriptional repressor of the master regulator of growth, Myc. AGO1 depletion in wing imaginal discs drives a significant increase in ribosome biogenesis, nucleolar expansion and cell growth in a manner dependent on Myc abundance. Moreover, increased Myc promoter activity and elevated Myc mRNA in AGO1-depleted animals requires RNA polymerase II transcription. Further support for transcriptional AGO1 functions is provided by physical interaction with the RNA polymerase II transcriptional machinery (chromatin remodelling factors and Mediator Complex), punctate nuclear localisation in euchromatic regions and overlap with Polycomb Group transcriptional silencing loci. Moreover, significant AGO1 enrichment is observed on the Myc promoter and AGO1 interacts with the Myc transcriptional activator Psi. Together, our data show that Drosophila AGO1 functions outside of the RISC to repress Myc transcription and inhibit developmental cell and tissue growth.This article has an associated 'The people behind the papers' interview.

Increased rates of ribosome biogenesis and biomass accumulation are fundamental properties of rapidly growing and dividing malignant cells. The MYC oncoprotein drives growth predominantly via its ability to upregulate the ribosome biogenesis program, in particular stimulating the activity of the RNA Polymerase I (Pol I) machinery to increase ribosomal RNA (rRNA) transcription. Although MYC function is known to be highly dependent on the cellular signalling context, the pathways interacting with MYC to regulate transcription of ribosomal genes (rDNA) in vivo in response to growth factor status, nutrient availability and cellular stress are only beginning to be understood. To determine factors critical to MYC-dependent stimulation of rDNA transcription in vivo, we performed a transient expression screen for known oncogenic signalling pathways in Drosophila. Strikingly, from the broad range of pathways tested, we found that ribosomal protein S6 Kinase (S6K) activity, downstream of the TOR pathway, was the only factor rate-limiting for the rapid induction of rDNA transcription due to transiently increased MYC. Further, we demonstrated that one of the mechanism(s) by which MYC and S6K cooperate is through coordinate activation of the essential Pol I transcription initiation factor TIF-1A (RRN 3). As Pol I targeted therapy is now in phase 1 clinical trials in patients with haematological malignancies, including those driven by MYC, these data suggest that therapies dually targeting Pol I transcription and S6K activity may be effective in treating MYC-driven tumours.

The ribosome is critical for all aspects of cell growth due to its essential role in protein synthesis. Paradoxically, many Ribosomal proteins (Rps) act as tumour suppressors in Drosophila and vertebrates. To examine how reductions in Rps could lead to tissue overgrowth, we took advantage of the observation that an RpS6 mutant dominantly suppresses the small rough eye phenotype in a cyclin E hypomorphic mutant (cycE(JP)). We demonstrated that the suppression of cycE(JP) by the RpS6 mutant is not a consequence of restoring CycE protein levels or activity in the eye imaginal tissue. Rather, the use of UAS-RpS6 RNAi transgenics revealed that the suppression of cycE(JP) is exerted via a mechanism extrinsic to the eye, whereby reduced Rp levels in the prothoracic gland decreases the activity of ecdysone, the steroid hormone, delaying developmental timing and hence allowing time for tissue and organ overgrowth. These data provide for the first time a rationale to explain the counter-intuitive organ overgrowth phenotypes observed for certain members of the Minute class of Drosophila Rp mutants. They also demonstrate how Rp mutants can affect growth and development cell non-autonomously.

Ecdysone triggers transcriptional changes via the ecdysone receptor (EcR) to coordinate developmental programs of apoptosis, cell cycle and differentiation. Data suggests EcR affects cell cycle gene expression indirectly and here we identify Wingless as an intermediary factor linking EcR to cell cycle.

The first characterised FUSE Binding Protein family member, FUBP1, binds single-stranded DNA to activate MYC transcription. Psi, the sole FUBP protein in Drosophila, binds RNA to regulate P-element and mRNA splicing. Our previous work revealed pro-growth functions for Psi, which depend, in part, on transcriptional activation of Myc. Genome-wide functions for FUBP family proteins in transcriptional control remain obscure. Here, through the first genome-wide binding and expression profiles obtained for a FUBP family protein, we demonstrate that, in addition to being required to activate Myc to promote cell growth, Psi also directly binds and activates stg to couple growth and cell division. Thus, Psi knockdown results in reduced cell division in the wing imaginal disc. In addition to activating these pro-proliferative targets, Psi directly represses transcription of the growth inhibitor tolkin (tok, a metallopeptidase implicated in TGFβ signalling). We further demonstrate tok overexpression inhibits proliferation, while tok loss of function increases mitosis alone and suppresses impaired cell division caused by Psi knockdown. Thus, Psi orchestrates growth through concurrent transcriptional activation of the pro-proliferative genes Myc and stg, in combination with repression of the growth inhibitor tok.

Despite two decades of research, the major function of FBP-family KH domain proteins during animal development remains controversial. The literature is divided between RNA processing and transcriptional functions for these single stranded nucleic acid binding proteins. Using Drosophila, where the three mammalian FBP proteins (FBP1-3) are represented by one ortholog, Psi, we demonstrate the primary developmental role is control of cell and tissue growth. Co-IP-mass spectrometry positioned Psi in an interactome predominantly comprised of RNA Polymerase II (RNA Pol II) transcriptional machinery and we demonstrate Psi is a potent transcriptional activator. The most striking interaction was between Psi and the transcriptional mediator (MED) complex, a known sensor of signaling inputs. Moreover, genetic manipulation of MED activity modified Psi-dependent growth, which suggests Psi interacts with MED to integrate developmental growth signals. Our data suggest the key target of the Psi/MED network in controlling developmentally regulated tissue growth is the transcription factor MYC. As FBP1 has been implicated in controlling expression of the MYC oncogene, we predict interaction between MED and FBP1 might also have implications for cancer initiation and progression.

Transcription of the ribosomal RNA genes (rDNA) by RNA polymerase I (Pol I) is a major control step for ribosome synthesis and is tightly linked to cellular growth. However, the question of whether this process is modulated primarily at the level of transcription initiation or elongation is controversial. Studies in markedly different cell types have identified either initiation or elongation as the major control point. In this study, we have re-examined this question in NIH3T3 fibroblasts using a combination of metabolic labeling of the 47S rRNA, chromatin immunoprecipitation analysis of Pol I and overexpression of the transcription initiation factor Rrn3. Acute manipulation of growth factor levels altered rRNA synthesis rates over 8-fold without changing Pol I loading onto the rDNA. In fact, robust changes in Pol I loading were only observed under conditions where inhibition of rDNA transcription was associated with chronic serum starvation or cell cycle arrest. Overexpression of the transcription initiation factor Rrn3 increased loading of Pol I on the rDNA but failed to enhance rRNA synthesis in either serum starved, serum treated or G0/G1 arrested cells. Together these data suggest that transcription elongation is rate limiting for rRNA synthesis. We propose that transcription initiation is required for rDNA transcription in response to cell cycle cues, whereas elongation controls the dynamic range of rRNA synthesis output in response to acute growth factor modulation.

Intronic polymorphic TOMM40 variants increasing TOMM40 mRNA expression are strongly correlated to late onset Alzheimer's Disease. The gene product, hTomm40, encoded in the APOE gene cluster, is a core component of TOM, the translocase that imports nascent proteins across the mitochondrial outer membrane. We used Drosophila melanogaster eyes as an in vivo model to investigate the relationship between elevated Tom40 (the Drosophila homologue of hTomm40) expression and neurodegeneration. Here we provide evidence that an overabundance of Tom40 in mitochondria invokes caspase-dependent cell death in a dose-dependent manner, leading to degeneration of the primarily neuronal eye tissue. Degeneration is contingent on the availability of co-assembling TOM components, indicating that an increase in assembled TOM is the factor that triggers apoptosis and degeneration in a neural setting. Eye death is not contingent on inner membrane translocase components, suggesting it is unlikely to be a direct consequence of impaired import. Another effect of heightened Tom40 expression is upregulation and co-association of a mitochondrial oxidative stress biomarker, DmHsp22, implicated in extension of lifespan, providing new insight into the balance between cell survival and death. Activation of regulated death pathways, culminating in eye degeneration, suggests a possible causal route from TOMM40 polymorphisms to neurodegenerative disease.

In both Drosophila melanogaster and mammalian systems, epithelial structure and underlying cell polarity are essential for proper tissue morphogenesis and organ growth. Cell polarity interfaces with multiple cellular processes that are regulated by the phosphorylation status of large protein networks. To gain insight into the molecular mechanisms that coordinate cell polarity with tissue growth, we screened a boutique collection of RNAi stocks targeting the kinome for their capacity to modify Drosophila "cell polarity" eye and wing phenotypes. Initially, we identified kinase or phosphatase genes whose depletion modified adult eye phenotypes associated with the manipulation of cell polarity complexes (via overexpression of Crb or aPKC). We next conducted a secondary screen to test whether these cell polarity modifiers altered tissue overgrowth associated with depletion of Lgl in the wing. These screens identified Hippo, Jun kinase (JNK), and Notch signaling pathways, previously linked to cell polarity regulation of tissue growth. Furthermore, novel pathways not previously connected to cell polarity regulation of tissue growth were identified, including Wingless (Wg/Wnt), Ras, and lipid/Phospho-inositol-3-kinase (PI3K) signaling pathways. Additionally, we demonstrated that the "nutrient sensing" kinases Salt Inducible Kinase 2 and 3 (SIK2 and 3) are potent modifiers of cell polarity phenotypes and regulators of tissue growth. Overall, our screen has revealed novel cell polarity-interacting kinases and phosphatases that affect tissue growth, providing a platform for investigating molecular mechanisms coordinating cell polarity and tissue growth during development.